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Conclusions Techniques for primary segmental epithelial culture and the expression of segmental nephron functions in culture, as presented, lend strong support to the notion that this "reductionistic" approach to the kidney has, by now, been instituted. The tacit assumption that "primaries" of segmental nephron populations are difficult to set up and "dedifferentiate" rapidly can no longer be regarded as justified. One may envision that cell lines derived from defined cultured nephron populations, in contrast to renal cell lines of nondefined origin, maintain the genotypic stability and phenotypic expression of their in vivo segment. In the final evaluation of this issue, without exposing improper subjectivity, the recent remarkable work on intracellular signaling systems and cyptoplasmic membrane ion channel activity in primary segmental cultures justifies the opinion that the interdependence of particular properties in a given nephron cell type may be expressed best in primary cultures.
[24] T i s s u e C u l t u r e o f E s t a b l i s h e d R e n a l C e l l L i n e s By N. L. SIMMONS Introduction The existence of several established epithelial cell lines derived from whole kidneys, which retain significant histotypic features, has ensured their popularity as general rather than specific models for studies of epithelial polarity, ion transport, and hormonal regulation. The most frequently used cell tines are MDCK (derived from a cocker spaniel kidney by Madin and Darby I and LLCPKI (derived from a hog kidney by Hull et al.2). There is continued interest in defining renal cell lines that may display useful properties, e.g., PK l and JTC cells. Established cell lines derived initially from human renal carcinomas (Cur, Caki-1) have received relatively tittle attention) An important caveat to the use of all established cell lines is to be aware of the possibility of phenotypic heterogeneity, not least from the origin of whole kidney enzymatic digests of most of the existing cell lines, but also from the existence of selection pressures arising from unique culture conI C. R. Gausch, W. L. Hard, and T. F. Smith, Proc. Soc. Exp. Biol. Med. 122, 931 (1966). 2 R. N. Hull, W. R. Cherry, and G. W. Weaver, In Vitro 12, 670 (1976). 3 N. F. Nelson, W. Cieplak, S. C. Dacus, and M. D. Prager, J. Cell. Physiol. 126, 435 (1986).
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ditions in individual laboratories. The use of clonal cell populations, with recloning to ensure homogeneity and stability of the phenotype of interest, is to be recommended. Even with cloned cell populations the observed phenotype may not be truly representative of any particular nephron segment or renal cell type and, indeed, the responsiveness to renal acting hormones in both the MDCK 4 and LLCPK~ cell lines5 is neither typical of cortical collecting tubules nor proximal tubule, respectively. In this chapter I will describe techniques and procedures for the routine culture and generation of epithelial layers used in our laboratory for MDCK cells. Stock Cultures Stock cultures of MDCK cells are grown in 75-cm2 glass (or plastic) tissue culture flasks in medium consisting of minimum essential Eagle's medium (MEM) supplemented with 2 m M glutamine, nonessential amino acids, and 5% (v/v) each of fetal calf serum and donor horse serum at 37 ° with kanamycin antibiotic (1 /zg/cm3), in an air/5% CO2 atmosphere. Antibiotic is usually included, but the use of antibiotic-free medium is preferable, if only to promote good aseptic practices. The exact concentration of fetal serum used is varied, but the minimum quantity is used that supports adequate growth. It is also a good practice to heat inactivate serum (complement) at 60 ° for 20 rain. A defined serum-free medium has been formulated for the MDCK cell line by Taub et al. 6 and contains Dulbecco's modified Eagle's medium: Ham's F12 [50% (v/v) each], 5 /zg/cm3 insulin, 5 #g/cm 3 transferrin, 50 n M hydrocortisone, 5 pM triiodothyronine, 0.1 /134 prostaglandin El, l0 nM selenium dioxide, and 1.1 g/liter NaHCO3/10 m M N-2-hydroxycthylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer. The general utility of this medium for the differing phenotypes displayed has not been established and our experience is that the growth rate declines after five to six passages. Cultures are seeded at a split ratio of 1:10. From a 75-cm 2 flask between 5-7.2 X l07 cells may be harvested in 7 days with a single medium change at day 3. Tissue culture flasks are placed on top of a thick glass plate to ensure minimal temperature variation in the constant-temperature cabinet/hot room 7 since similar patterns to those observed in LLCPKI cells were observed in MDCK 4 K. E. Meier and P. A. Insel, in "Tissue Culture of Epithelial Ceils" (M. Taub, ed.), p. 145. Plenum, New York, 1985. 5 A. Wohlwend, D. B. Vassalli, and L. Orci, Am. J. Physiol. 250, C682 (1986). 6 M. Taub, L. Chuman, M. H. Saier, and G. Sato, Proc. Natl. Acad. Sci. U.S.A. 76, 3338 (1979). 7 E. M. Adler, L. J. Fluk, J. M. Mullin, andA. Kleinzeller, Science217, 851 (1982).
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cultures. The number of serial passages is limited (typically 15) and frequent recourse is made to the frozen stocks (see Freezing of Cell Stocks, below). Since the importance of "bottom feeding" in the maintenance of epithelial characteristics is now generally recognized for some cell lines,8 the use of impermeant supports could be questioned. Since significant transjunctional permeability of nonelectrolytes in leaky epithelia has been documented, this may not be a serious problem in some cultures. If a serious problem is envisaged by the use ofimpermeant supports, restriction of seeding densities and growth times to ensure subeonfluent conditions should be considered. Formation of a single-cell suspension for serial passage of stock tissue culture flasks is made by washing the cell monolayer three times in a Ca2+-Mg2+-free Dulbecco's salt solution, followed by incubation in 5 cm 3 of 0.25% trypsin-2 m M ethylenediaminetetraacetic acid (EDTA) in Ca2+-Mg2+-free Earle's basal salt solution at 37 °, until the complete detachment of the cell monolayer is achieved. With MDCK cells particular importance is attached to this step, as incomplete harvesting of the cells would result in an inherent selection pressure. A single-cell suspension is obtained by gentle syringing through a wide-bore needle (2-mm i.d.). Trypsin is neutralized by the addition of complete growth medium and cell density determined directly by electronic cell counting and sizing of an aliquot of the cell suspension diluted in Isoton (Coulter Electronics Ltd., Luton, England)? We presently buy all tissue culture stocks from Gibco (Paisley, Scotland) though in the past we have obtained supplies from How laboratories (Rickmansworth, England). The dependence of the researcher on the high quality of sterile products produced commercially does not need to be emphasized. Preparation of Cell Clones I have already stressed the importance of the use of cell clones that express stable phenotypic properties. Table I demonstrates the measured phenotype of two extensively used and characterized clonal cell lines of MDCK. Also, the properties of MDCK cells from different sources are shown. The following procedure, presently used in our laboratory for cloning MDCK cells, is from a method outlined by K. Soderberg at European Molecular Biology Laboratory (Heidelberg, West Germany). A monodisperse cell suspension is made from a logarithmically growing 8j. s. Handler,A. S. Preston,and R. E. Steele,Fed. Proc., Fed. Am. Soc. Exp. Biol. 43, 2221 (1984). 9 L. Boardman, M. Huett, J. F. Lamb, J. P. Newton, and J. Poison, J. Physiol. (London) 241, 771 (1974).
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TABLE I VARIATION IN THE STABLE PHENOTYPIC CHARACTERISTICS BETWEEN DIFFERENT MDCK CELL STRAINS AND CELL CLONESa
MDCK cell
Strain I b Strain 2b Clone DL~7
Clone CL8~b MDCKNI
Diuretic-sensitive K + transport [nmol/( 106 ceils, rain)]
Transepithelial resistance (~-em 2)
8.8 + 2.6 NDc NDc 3.9 + 0.7 1.8 + 0.4
2500-7000 100 (cation)a 100 (cation)a 2500-4000 1500-2500
Adenylate cyclase (-fold stimulation over basal)
2.85 + 1.07 + 1.03 + 1.50 + 1.80 +
0.24 0.04 0.08 0.05 0.03
a With respect to some of the measured parameters describedin the text. Diuretic-sensitivetransport is that portion of the ouabain-insensitiveflux that is inhibited by 0.1 ~ loop diuretic (furosemide, bumetanide). Transepithelial resistance was determined from epithelial layers grown on uncoated Millipore filters which were then damped into Ussing chambers. Isoprenaline (100/zM)-stimulated adenylate cyclaseactivity was determined in disrupted cells in the presence oflBMX. b From G. Barker and N. L. Simmons, Q. J. Exp. Physiol. 66, 61 (1981). c ND, Not detectable. a Cation refers to the existenceof a cation-selectiveparaceilular pathway. e From E. L. Rugg and N. L. Simmons, Q. J. Exp. Physiol. 69, 405 (1984). I From J. A. McRoberts, C. Trong-Trang, and M. H. Saier, J. Biol. Chem. 258, 12320 (1983). stock culture, as outlined above. Typically 1000 cells are then plated directly onto a 10-cm diameter Petri dish ( N u n c plastic, Gibco, Paisley, Scotland). The growth m e d i u m is enriched by the use o f 20% fetal calf serum. Plates are then incubated ~ r approximately 14 days until single-cell colonies are visible ( 2 - 3 m m in diameter). The plates are then washed in Ca2+-Mg2+-free Earle's salt solution and sterile cloning cylinders (5 m m long, 7 - m m o.d, 6 - m m i.d.) are placed a r o u n d isolated colonies by sealing them to the culture dish with autoclaved high-viscosity v a c u u m grease. The individual colony is then trypsinized as above and the resulting cell suspension transferred to 1 well o f a 12-well plate for further expansion. A second cloning ensures the clonal nature o f the cell culture. Time-lapse photomicroscopy o f subconfluent cultures has demonstrated significant cellular motility and cell aggregation. An appropriate cell density for formation o f single-cell colonies is therefore critical. F r e e z i n g o f Cell S t o c k s All cell stocks are maintained frozen above liquid nitrogen. Concentrated cell suspensions (to 1 X 106 cells/cm 3) are made by centrifugation (250 g) and resuspension in an appropriate volume o f complete culture m e d i u m supplemented with 10% (v/v) dimethylsulfoxide (DMSO) as cryoprotectant. Freezing o f cultures should be performed so that there is a
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controlled initial reduction (no greater than - 5 ° / m i n ) to obviate the formation of large ice crystals. We achieve this by placing the vials (5 em 3 total capacity), wrapped in cotton wool, in an expanded polystyrene carton and placing this in a - 9 0 ° cabinet overnight. Final equilibration to liquid nitrogen temperature may then be achieved rapidly. On thawing, vials are directly incubated in a shaking water bath held at 37 °. It should be noted that different epithelial parameters have recently been reported for a single batch of MDCK cells held in aliquots, frozen, thawed, and recultured. 1° Detection of Mycoplasma Contamination Mycoplasma is a common contaminant of established cell lines. Routine detection of such contamination is detected by DNA fluorochromes such as bisbenzimide (Hoechst 33258, Aldrich Chemical Co. Ltd., Gillingham, England), which allows the detection of mycoplasma. H Hoe 33258 (5 rag) is dissolved in 100 cm 3 of Ca2+-Mg2+-free phosphate-buffered saline (PBS) and stored in the dark at 4 °. Cell monolayers seeded directly onto glass coverslips [or Millipore (Bedford, MA) filter supports] are rinsed in PBS and then fixed in 3:1 (v/v) methanol/acetic acid for 10 min and then air dried. The cell layers are then stained with a 1:100 dilution of the stock Hoe 33258 for 10 min, washed in distilled water, and wet mounted for observation under incident light epifluorescence (UV excitation, LP 420 barrier filters; Zeiss, Oberkochen, FRG). Elimination of mycoplasma from established cell lines can be attempted using cell cloning or repeated incubation with agents such as BMcycline (Cat. No. 799050; Boehringer-Mannheim, Mannheim, FRG). Measurements of T r a n s m e m b r a n e Solute Transport Measurements of plasma membrane solute transport or receptor density may be made directly using radiotracers in cells cultivated on small 3-cm dishes or in 6-, 12-, or 96-well dishes, depending on particular requirements imposed by the specific activities of the solute of interest and the capacity or density of membrane transport sites or receptor density present on individual cultures. Specific attention should be focused on the cellular location of the system of interest, as access to the basolateral surfaces may be considerably restricted in confluent cultures. Even in subconfluent cultures autoradiography has demonstrated the restriction of cellular uptake of certain amino acids to cells on the periphery of cell ,o R. F. Husted, M. J. Welsh, and J. B. Stokes, Am. J. Physiol. 250, C214 (1986). " T. R. Chen, Exp. CellRess. 104, 255 (1977).
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islands in LLCPK~ cells. '2 In any case the density may be an important consideration, ff high cellular densities are required for the expression of transport function. An alternative approach would be to ensure disruption of the apical tight junction using incubations in Ca2+-Mg2+-free medium containing EDTA, '3 or to perform the experiments on cells in suspension. 12 Adequate controls must then be performed to demonstrate that these procedures do not interfere with the membrane transport of interest. For MDCK cells where K + transport (unidirectional influx) is measured using the K + congener SrRb under equilibrium exchange conditions we aim to produce subconfluent layers after 3 days of culture. A seeding density of 5 × 104 cells/cm2 is used to realize a final density for experiments of 1.5-2.0 × 105 cells/cm2. All experimental incubations are performed in nonsterile conditions on a bench incubator made of an aluminum trough supported on a Perspex plate through which thermostated water is forced by a flow heater (Churchill Instrument Co. Ltd., Uxbridge, England). This necessitates the use of phosphate, Tris, or HEPES-buffered experimental medium. The usual composition of the experimental buffer is 137 m M NaC1, 5.4 m M KC1, 2.8 m M CaC12, 1.2 m M MgSO4, 0.3 m M NaH2PO4, 0.3 m M KH2PO4, 14 m M Tris, 12 m M HC1, pH 7.4, at 37 °. A preincubation step of 20 min usually precedes flux determinations, due to a transiently elevated flux upon transfer from the growth medium to the experimental medium. Glucose (5 mM) and donor horse serum [1% (v/v) or lower] are the only additions required to maintain cell viability for prolonged periods (up to 3 hr). The flux measurement is initiated by the addition of the prewarmed radiotracer-containing solution (3..7 kBq/cm 3 SrRb; Amersham International, Amersham, England), and terminated by its rapid removal by suction via a water-driven pump and by repeated washing (five or six times, - 3 0 sec each time) in ice-cold tracer-free wash medium (usually the incubation medium). With cultured cells on plastic such washes achieve extremely rapid temperature reductions (~ 100 msec). Artifactual loss of isotope is thus low, but may be reduced by inclusion of transport inhibitors in the "stopping solution," e.g., HgCI2 for facilitated sugar transport. The adequacy of the wash protocol must be checked by the efficient clearance of an extracellular marker such as [~4C]- or [3H]inulin. The total time for measurement of undirectional uptake requires that the flux is undertaken in the linear portion of the uptake curve. For ~Rb a 5- or 10-rain influx period is usual. For other ions, e.g., 22 Na, extremely rapid influx determinations must be used (10 see); this is not easily performed since high ,2 F. V. Sepulveda and J. D. Pearson, J. Cell. Physiol. 118, 211 (1984). ,3 K. Matlin, D. F. Bainton, M. Pesonen, D. Louvard, N. Genty, K. Simons, J. CellBiol. 97, 627 0983).
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isotope-specific activities need to be used. It should be noted that determinations of influx of Na + in MDCK cells in some studies are made under conditions of net uptake in Na+-depleted cells. 14 Flux determinations are normalized to cell number/plate (most usual), protein, or DNA content using an aliquot of the extracted well. If flux is normalized by cell number determinations extraction of isotope is made with a trypsin- 2 m M EDTA solution to form a single-cell suspension as outlined above. An important control that is always performed is to test for nonspecific uptake of radiotracer on plastic Petri dishes in the absence of seeded cells. This is especially important for binding determinations. Unidirectional efflux is performed using a single-plate technique with continual replacement (every 3 min) of the external medium. This necessitates the preequilibration of the intracellular fluid with S6Rb by incubation in experimental medium containing 37 kBq/cm 3 S6Rb for 3 hr. Rate coefficients for S6Rb loss can be calculated from semilog plots of S6Rb loss against time. Measurements of net ion movements and of total ionic composition are made by washing the cell layers in ice-cold wash medium (usually isotonic choline chloride for Na + and K + determinations) and by final extraction in double glass-distilled water for 3 - 4 hr. Sodium ion and K + are directly determined using flame photometry. Determination of cell volumes in monolayer cultures is most conveniently measured using the technique of Kletzein et al? 5 Cell layers are incubated to equilibrium (60 rain) with 37 kBq/cm 3 of the nonmetabolizable sugar 3-O-methyl-D-[~4C]glucose as an intracellular space marker. It is important to check that no uphill transport occurs (as would occur in LLCPK t cells) and that there is no intracellular binding. Washing and extraction is carried out as described for K + fluxes. If the demonstration of the epithelial polarity of transport or binding is required, influx, etflux, and binding determinations are possible on epithelial layers grown upon permeant supports, using techniques identical in principle to those described. The filter support in this case will impose a serious "unstirred layer" for measurements across the basal surfaces. Preparation of Epithelial L a y e r s on P e r m e a n t Supports Uncoated Millipore (MF) Supports
Epithelial layers of MDCK cells have been successfully generated with pore sizes up to 2/~m. Generally the larger the pore size the slower the ~4M. J. Kindler,M. Taub, and M. H. Saier,J. CellBiol. 254, 11431 (1979). ~5R. F. Kletzein,M. W. Pariza,J. E. Becker,and V. R. Potter,Anal. Biochem. 69, 537 (1975).
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attainment of a confluent layer. We have not successfully generated epithelial layers in larger pore sizes without coating the support with collagen. Seeding is carried out by directly applying the cell suspension at high cell densities (2 × 105 cells/cm2 of permeable support) onto Millipore filters (2.5-cm diameter) (boiled in 100 cm 3 distilled water to remove the wetting agent) and held in presterilized Swinnex holders (Millipore Ltd., Harrow, England). The holder is incubated at 37* in an air/5% CO2 atmosphere for up to 1 hr to allow cell attachment. After this time the filter is removed from the holder, the layer is washed free of nonadherent cells, and the layer is finally floated in complete medium to which insulin is invariably added. After 3 days confluent epithelial layers are formed and we usually perform our experiments at this time. However, the medium may be changed every 3 days and the epithelial layers maintained. For routine experiments involving measurement of epithelial parameters we favor this method, as the epithelial layers may then be clamped directly into Ussing chambers in which edge effects are reduced in a conventional way. It should be stressed that edge effects exist even with epithelia grown in mini-Marbrook-type chambers, as epithelial cells are unlikely to form functional junctions with the vertical walls of the chamber. The morphology of MDCK cells certainly varies toward the outer edge of the cup under such conditions.
Collagen-CoatedMillipore Filters Collagen (5 mg/cm 3) [made from rat tails or purchased from Sigma (St. Louis, MO)] in 1% acetic acid is poured directly onto 2.5-cm Millipore filter disks contained in a 10-cm Petri dish and spread to give an even coverage. Gelation is achieved by exposure to ammonia vapor from 1 to 2 cm 3 of ammonia solution. The ammonia solution is removed and the filters allowed to dry. The collagen gel is then cross-linked by immersion in 4% glutaraldehyde solution for 60 min. Repeated washing with MEM ensures removal of any residual giutaraldehyde. Cell seeding is achieved by placing the collagen-coated filters in small (3 cm) dishes and proceeding as above.
Assessment of Confluency on Millipore Substrates Assessment of confluency in cell layers grown on Millipore filters is made by a conventional fixation (2% glutaraldehyde, staining with hemotoxylin) and dehydration schedule. Filters in absolute alcohol are then cleared in a 50:50 mixture of absolute alcohol and toluene, and finally in toluene. The filters are air dried and mounted on microscope slides in Canada balsam, which, since it has the same refractive index as the filter, renders it translucent. The epithelial layer may then be visualized directly.
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Growth of Epithelial Layers on Collagen-Coated Nylon Mesh Collagen gel may be used to form a translucent substrate in a nylon mesh support, as initially described by Cereijido et al. t6 Disks of nylon mesh (2.5 era; Simonyl N118, Henry Simon Textiles, Stockport Cheshire, England) are first cleaned by sonication in acetone (5 min), boiling in distilled water (10 min), and then washing in an ascending series of alcohols before being dried. They are then dipped in acetic acid-collagen solution and processed as are Millipore filters. The strength of the collagen gel was often sufficient such that the glutaraldehyde cross-linking step could be omitted. The omission of the glutaraldehyde fixation step facilitated microelectrode penetration through the collagen matrix. Final sterilization of the collagen/nylon substrates was achieved by short-wave UV irradiation (30 min). Storage of both collagen-covered Millipore filters and nylon/collagen matrices was possible in MEM for up to 1 month at 4". An alternative translucent substrate based on chitosan has been developed and shown to be perfectly adequate for the formation of MDCK epithelia.22 Growth in Marbrook- Type Chambers A variety of arrangements have been adopted to allow direct culture of epithelial layers in which separate access to each epithelial surface is achieved. Our initial method was to clamp Millipore filters directly into commercially available (Hendley Engineering Co., Loughton, Essex, England) nylon mini-Marbrook chambers. 17 These chambers consist of two portions to which the filter directly clips. They may be sterilized preassembled and have small feet to allow direct access of medium to the basal surfaces of the developing epithelium. Seeding is achieved by direct addition of the concentrated cell suspension to the chamber outlined above, except that fresh growth medium is finally added to both the upper chamber ( 1.5 cm a) and the 3-cm Petri (3 cm a) dish in which the mini-Marbrook chamber is placed. During growth the upper chamber often becomes acidified before the lower chamber, indicating functional (H +) transport. The great advantage of the mini-Marbrook arrangement is for longer term experiments in which epithelial cultures can be monitored continually using salt bridges cleaned by alcohol wipes, and for experiments in which access to both epithelial surfaces is required for collection of secreted products, for biochemical labeling experiments, or for transepithelial cell migration studies. 16 M. Cereijido, E. S. Robbins, W. J. Dolan, C. A. Rotunno, and D. D. Sabatini, J. Cell Biol. 77, 853 (1978). '~ J. C. W. Richardson and N. L. Simmons, FEBSLett. 105, 201 (1979).
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More recently several different designs have been introduced encompassing similar design features to those existing in the commercial miniMarbrook chambers.~8 In addition, different sizes of presterilized chambers consisting of membrane filter glued to a polystyrene body are now available [e.g., Millicell-HA; a transparent membrane filter (Biopore, MillicellCM) allows direct microscopy of growing epithelial layers. Such transparent membranes need to be precoated with a biological matrix such as collagen; our own experience is that lower epithelial resistance values are achieved with Millicell-CM compared with Millicell-HA.] A novel adaptation of this idea was developed by the Simons group in Heidelburg. ~9The Millipore millititer HA 96-well plate for microfiltration can be used to produce a large number of identical epithelial layers suitable for rapid screening of the effects of agents such as monoclonal antibodies (or other bioactive reagents) on epithelial junction formation. The impermeable backing is first stripped from the 96-well plate, and the sterile packaging bubble can be used as the bottom chamber. Cell seeding is achieved, exactly as described above. Our experience is that entirely adequate epithelial layers may be generated, though the electrical resistance is of a somewhat lower magnitude. Measurement of Transepithelial Electrical Properties (Resistance, Short-Circuit Current, Dilution, and Biionic Potential Differences (pd) A detailed and basic discussion of this topic, including construction, calibration, and use of reversible electrodes and salt bridges, is given in an excellent review by Watlington et al. 2° As previously mentioned, epithelial layers may be mounted directly using large areas and silicone seals to reduce edge effects, 21 o r whole mini-Marbrook chambers may be clamped directly into specially adapted chambers. For the purpose of the present chapter it is worth stressing the importance of the correct geometry needed with respect to the measuring and current-passing Ringer bridges, when applied to the p.d. measurement of the electrical resistance of "leaky" epithelial layers, and of the necessity of making an appropriate correction for the series resistance afforded by the solution resistor present between the potential-sensing electrodes. This point is also of practical importance 18 R. E. Steele, A. S. Preston, J. P. Johnson, and J. S. Handler, Am. J. Physiol. 251, C136 0986). ,9 D. Gumbiner and K. Simons, J. Cell Biol. 102, 457 0986). 2o C. O. Watlington, J. C. Smith, and E. G. Huf, Exp. Physiol. Biochem. 3, 49 (1970). 21 N. L. Simmons, C. D. A. Brown, and E. L. Rug& Fed. Proc., Fed. Am. Soc. Exp. Biol. 43, 2225 0984).
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when short circuiting the epithelial layer. In standard Ussing chambers made of Perspex, drilled guide holes for the potential and current-passing agar bridges are present, ensuring that no change in the distance between the potential-sensing electrodes occurs on chamber assembly/disassembly, and that the current-passing bridges are placed sufficiently distant from the epithelial layer to ensure an even current density (the rule of thumb is two times the radius of the epithelial layer). For accurate determination of epithelial properties, it is simply not sufficient to dip the agar bridges in the bathing solutions, though this may be adequate for rapid screening. Often the DC electrical resistance of the epithelium might not be the most convenient or reliable method for determining the functional presence of an epithelial layer. If a significant paracellular pathway for ions is present with ionic mobilities differing from free solution, a more convenient method is to elicit a dilution or biionic pd. For MDCK epithelial layers this can be achieved by replacing the basal bathing solution for one in which the NaC1 is replaced in an equimolar fashion by choline chloride) 6 The resulting concentration gradient for Na + and the cationic permselectivity of the paracellular pathway will render the basal bathing solution up to 45 m V electropositive with respect to the apical bathing solution. Replacement of the basal bathing solution is recommended as the epithelial layer is less subject to damage from solution turbulence under these conditions. M e a s u r e m e n t of Bidirectional Transepithelial Solute Fluxes Simultaneous measurement of bidirectional transepitbelial fluxes using 3H-or 14C-labeled solutes or 22Na and UNa is recommended whenever possible in order to eliminate time-dependent variation and betweenmonolayer variation. Isotopes are added to both bathing solutions (volume = 8 crn 3) and after an initial 30-rain flux period, 1-cm 3 samples of each chamber are taken. Two further 1-cm 3 samples at 60 and 90 rain are taken and fluxes averaged over these two 30-rain periods. Flux is calculated from the rate of tracer appearance on the contralateral side, to which radiotracer was added. Isotope equilibration should not exceed 1 -2%, and the specific activities used should allow increments of 5 X 102 Bq/cm 3 in the contralateral chamber.
22 p. Popowicz, J. Kurzyca, B. Dolinska, and J. Popowicz, Biomed. Biochim. Acta 44, 1329 (1985)
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Conclusions Techniques for primary segmental epithelial culture and the expression of segmental nephron functions in culture, as presented, lend strong support to the notion that this "reductionistic" approach to the kidney has, by now, been instituted. The tacit assumption that "primaries" of segmental nephron populations are difficult to set up and "dedifferentiate" rapidly can no longer be regarded as justified. One may envision that cell lines derived from defined cultured nephron populations, in contrast to renal cell lines of nondefined origin, maintain the genotypic stability and phenotypic expression of their in vivo segment. In the final evaluation of this issue, without exposing improper subjectivity, the recent remarkable work on intracellular signaling systems and cyptoplasmic membrane ion channel activity in primary segmental cultures justifies the opinion that the interdependence of particular properties in a given nephron cell type may be expressed best in primary cultures.
[24] T i s s u e C u l t u r e o f E s t a b l i s h e d R e n a l C e l l L i n e s By N. L. SIMMONS Introduction The existence of several established epithelial cell lines derived from whole kidneys, which retain significant histotypic features, has ensured their popularity as general rather than specific models for studies of epithelial polarity, ion transport, and hormonal regulation. The most frequently used cell tines are MDCK (derived from a cocker spaniel kidney by Madin and Darby I and LLCPKI (derived from a hog kidney by Hull et al.2). There is continued interest in defining renal cell lines that may display useful properties, e.g., PK l and JTC cells. Established cell lines derived initially from human renal carcinomas (Cur, Caki-1) have received relatively tittle attention) An important caveat to the use of all established cell lines is to be aware of the possibility of phenotypic heterogeneity, not least from the origin of whole kidney enzymatic digests of most of the existing cell lines, but also from the existence of selection pressures arising from unique culture conI C. R. Gausch, W. L. Hard, and T. F. Smith, Proc. Soc. Exp. Biol. Med. 122, 931 (1966). 2 R. N. Hull, W. R. Cherry, and G. W. Weaver, In Vitro 12, 670 (1976). 3 N. F. Nelson, W. Cieplak, S. C. Dacus, and M. D. Prager, J. Cell. Physiol. 126, 435 (1986).
METHODS IN ENZYMOLOGY,VOL. 191
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ditions in individual laboratories. The use of clonal cell populations, with recloning to ensure homogeneity and stability of the phenotype of interest, is to be recommended. Even with cloned cell populations the observed phenotype may not be truly representative of any particular nephron segment or renal cell type and, indeed, the responsiveness to renal acting hormones in both the MDCK 4 and LLCPK~ cell lines5 is neither typical of cortical collecting tubules nor proximal tubule, respectively. In this chapter I will describe techniques and procedures for the routine culture and generation of epithelial layers used in our laboratory for MDCK cells. Stock Cultures Stock cultures of MDCK cells are grown in 75-cm2 glass (or plastic) tissue culture flasks in medium consisting of minimum essential Eagle's medium (MEM) supplemented with 2 m M glutamine, nonessential amino acids, and 5% (v/v) each of fetal calf serum and donor horse serum at 37 ° with kanamycin antibiotic (1 /zg/cm3), in an air/5% CO2 atmosphere. Antibiotic is usually included, but the use of antibiotic-free medium is preferable, if only to promote good aseptic practices. The exact concentration of fetal serum used is varied, but the minimum quantity is used that supports adequate growth. It is also a good practice to heat inactivate serum (complement) at 60 ° for 20 rain. A defined serum-free medium has been formulated for the MDCK cell line by Taub et al. 6 and contains Dulbecco's modified Eagle's medium: Ham's F12 [50% (v/v) each], 5 /zg/cm3 insulin, 5 #g/cm 3 transferrin, 50 n M hydrocortisone, 5 pM triiodothyronine, 0.1 /134 prostaglandin El, l0 nM selenium dioxide, and 1.1 g/liter NaHCO3/10 m M N-2-hydroxycthylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer. The general utility of this medium for the differing phenotypes displayed has not been established and our experience is that the growth rate declines after five to six passages. Cultures are seeded at a split ratio of 1:10. From a 75-cm 2 flask between 5-7.2 X l07 cells may be harvested in 7 days with a single medium change at day 3. Tissue culture flasks are placed on top of a thick glass plate to ensure minimal temperature variation in the constant-temperature cabinet/hot room 7 since similar patterns to those observed in LLCPKI cells were observed in MDCK 4 K. E. Meier and P. A. Insel, in "Tissue Culture of Epithelial Ceils" (M. Taub, ed.), p. 145. Plenum, New York, 1985. 5 A. Wohlwend, D. B. Vassalli, and L. Orci, Am. J. Physiol. 250, C682 (1986). 6 M. Taub, L. Chuman, M. H. Saier, and G. Sato, Proc. Natl. Acad. Sci. U.S.A. 76, 3338 (1979). 7 E. M. Adler, L. J. Fluk, J. M. Mullin, andA. Kleinzeller, Science217, 851 (1982).
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cultures. The number of serial passages is limited (typically 15) and frequent recourse is made to the frozen stocks (see Freezing of Cell Stocks, below). Since the importance of "bottom feeding" in the maintenance of epithelial characteristics is now generally recognized for some cell lines,8 the use of impermeant supports could be questioned. Since significant transjunctional permeability of nonelectrolytes in leaky epithelia has been documented, this may not be a serious problem in some cultures. If a serious problem is envisaged by the use ofimpermeant supports, restriction of seeding densities and growth times to ensure subeonfluent conditions should be considered. Formation of a single-cell suspension for serial passage of stock tissue culture flasks is made by washing the cell monolayer three times in a Ca2+-Mg2+-free Dulbecco's salt solution, followed by incubation in 5 cm 3 of 0.25% trypsin-2 m M ethylenediaminetetraacetic acid (EDTA) in Ca2+-Mg2+-free Earle's basal salt solution at 37 °, until the complete detachment of the cell monolayer is achieved. With MDCK cells particular importance is attached to this step, as incomplete harvesting of the cells would result in an inherent selection pressure. A single-cell suspension is obtained by gentle syringing through a wide-bore needle (2-mm i.d.). Trypsin is neutralized by the addition of complete growth medium and cell density determined directly by electronic cell counting and sizing of an aliquot of the cell suspension diluted in Isoton (Coulter Electronics Ltd., Luton, England)? We presently buy all tissue culture stocks from Gibco (Paisley, Scotland) though in the past we have obtained supplies from How laboratories (Rickmansworth, England). The dependence of the researcher on the high quality of sterile products produced commercially does not need to be emphasized. Preparation of Cell Clones I have already stressed the importance of the use of cell clones that express stable phenotypic properties. Table I demonstrates the measured phenotype of two extensively used and characterized clonal cell lines of MDCK. Also, the properties of MDCK cells from different sources are shown. The following procedure, presently used in our laboratory for cloning MDCK cells, is from a method outlined by K. Soderberg at European Molecular Biology Laboratory (Heidelberg, West Germany). A monodisperse cell suspension is made from a logarithmically growing 8j. s. Handler,A. S. Preston,and R. E. Steele,Fed. Proc., Fed. Am. Soc. Exp. Biol. 43, 2221 (1984). 9 L. Boardman, M. Huett, J. F. Lamb, J. P. Newton, and J. Poison, J. Physiol. (London) 241, 771 (1974).
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TABLE I VARIATION IN THE STABLE PHENOTYPIC CHARACTERISTICS BETWEEN DIFFERENT MDCK CELL STRAINS AND CELL CLONESa
MDCK cell
Strain I b Strain 2b Clone DL~7
Clone CL8~b MDCKNI
Diuretic-sensitive K + transport [nmol/( 106 ceils, rain)]
Transepithelial resistance (~-em 2)
8.8 + 2.6 NDc NDc 3.9 + 0.7 1.8 + 0.4
2500-7000 100 (cation)a 100 (cation)a 2500-4000 1500-2500
Adenylate cyclase (-fold stimulation over basal)
2.85 + 1.07 + 1.03 + 1.50 + 1.80 +
0.24 0.04 0.08 0.05 0.03
a With respect to some of the measured parameters describedin the text. Diuretic-sensitivetransport is that portion of the ouabain-insensitiveflux that is inhibited by 0.1 ~ loop diuretic (furosemide, bumetanide). Transepithelial resistance was determined from epithelial layers grown on uncoated Millipore filters which were then damped into Ussing chambers. Isoprenaline (100/zM)-stimulated adenylate cyclaseactivity was determined in disrupted cells in the presence oflBMX. b From G. Barker and N. L. Simmons, Q. J. Exp. Physiol. 66, 61 (1981). c ND, Not detectable. a Cation refers to the existenceof a cation-selectiveparaceilular pathway. e From E. L. Rugg and N. L. Simmons, Q. J. Exp. Physiol. 69, 405 (1984). I From J. A. McRoberts, C. Trong-Trang, and M. H. Saier, J. Biol. Chem. 258, 12320 (1983). stock culture, as outlined above. Typically 1000 cells are then plated directly onto a 10-cm diameter Petri dish ( N u n c plastic, Gibco, Paisley, Scotland). The growth m e d i u m is enriched by the use o f 20% fetal calf serum. Plates are then incubated ~ r approximately 14 days until single-cell colonies are visible ( 2 - 3 m m in diameter). The plates are then washed in Ca2+-Mg2+-free Earle's salt solution and sterile cloning cylinders (5 m m long, 7 - m m o.d, 6 - m m i.d.) are placed a r o u n d isolated colonies by sealing them to the culture dish with autoclaved high-viscosity v a c u u m grease. The individual colony is then trypsinized as above and the resulting cell suspension transferred to 1 well o f a 12-well plate for further expansion. A second cloning ensures the clonal nature o f the cell culture. Time-lapse photomicroscopy o f subconfluent cultures has demonstrated significant cellular motility and cell aggregation. An appropriate cell density for formation o f single-cell colonies is therefore critical. F r e e z i n g o f Cell S t o c k s All cell stocks are maintained frozen above liquid nitrogen. Concentrated cell suspensions (to 1 X 106 cells/cm 3) are made by centrifugation (250 g) and resuspension in an appropriate volume o f complete culture m e d i u m supplemented with 10% (v/v) dimethylsulfoxide (DMSO) as cryoprotectant. Freezing o f cultures should be performed so that there is a
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controlled initial reduction (no greater than - 5 ° / m i n ) to obviate the formation of large ice crystals. We achieve this by placing the vials (5 em 3 total capacity), wrapped in cotton wool, in an expanded polystyrene carton and placing this in a - 9 0 ° cabinet overnight. Final equilibration to liquid nitrogen temperature may then be achieved rapidly. On thawing, vials are directly incubated in a shaking water bath held at 37 °. It should be noted that different epithelial parameters have recently been reported for a single batch of MDCK cells held in aliquots, frozen, thawed, and recultured. 1° Detection of Mycoplasma Contamination Mycoplasma is a common contaminant of established cell lines. Routine detection of such contamination is detected by DNA fluorochromes such as bisbenzimide (Hoechst 33258, Aldrich Chemical Co. Ltd., Gillingham, England), which allows the detection of mycoplasma. H Hoe 33258 (5 rag) is dissolved in 100 cm 3 of Ca2+-Mg2+-free phosphate-buffered saline (PBS) and stored in the dark at 4 °. Cell monolayers seeded directly onto glass coverslips [or Millipore (Bedford, MA) filter supports] are rinsed in PBS and then fixed in 3:1 (v/v) methanol/acetic acid for 10 min and then air dried. The cell layers are then stained with a 1:100 dilution of the stock Hoe 33258 for 10 min, washed in distilled water, and wet mounted for observation under incident light epifluorescence (UV excitation, LP 420 barrier filters; Zeiss, Oberkochen, FRG). Elimination of mycoplasma from established cell lines can be attempted using cell cloning or repeated incubation with agents such as BMcycline (Cat. No. 799050; Boehringer-Mannheim, Mannheim, FRG). Measurements of T r a n s m e m b r a n e Solute Transport Measurements of plasma membrane solute transport or receptor density may be made directly using radiotracers in cells cultivated on small 3-cm dishes or in 6-, 12-, or 96-well dishes, depending on particular requirements imposed by the specific activities of the solute of interest and the capacity or density of membrane transport sites or receptor density present on individual cultures. Specific attention should be focused on the cellular location of the system of interest, as access to the basolateral surfaces may be considerably restricted in confluent cultures. Even in subconfluent cultures autoradiography has demonstrated the restriction of cellular uptake of certain amino acids to cells on the periphery of cell ,o R. F. Husted, M. J. Welsh, and J. B. Stokes, Am. J. Physiol. 250, C214 (1986). " T. R. Chen, Exp. CellRess. 104, 255 (1977).
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islands in LLCPK~ cells. '2 In any case the density may be an important consideration, ff high cellular densities are required for the expression of transport function. An alternative approach would be to ensure disruption of the apical tight junction using incubations in Ca2+-Mg2+-free medium containing EDTA, '3 or to perform the experiments on cells in suspension. 12 Adequate controls must then be performed to demonstrate that these procedures do not interfere with the membrane transport of interest. For MDCK cells where K + transport (unidirectional influx) is measured using the K + congener SrRb under equilibrium exchange conditions we aim to produce subconfluent layers after 3 days of culture. A seeding density of 5 × 104 cells/cm2 is used to realize a final density for experiments of 1.5-2.0 × 105 cells/cm2. All experimental incubations are performed in nonsterile conditions on a bench incubator made of an aluminum trough supported on a Perspex plate through which thermostated water is forced by a flow heater (Churchill Instrument Co. Ltd., Uxbridge, England). This necessitates the use of phosphate, Tris, or HEPES-buffered experimental medium. The usual composition of the experimental buffer is 137 m M NaC1, 5.4 m M KC1, 2.8 m M CaC12, 1.2 m M MgSO4, 0.3 m M NaH2PO4, 0.3 m M KH2PO4, 14 m M Tris, 12 m M HC1, pH 7.4, at 37 °. A preincubation step of 20 min usually precedes flux determinations, due to a transiently elevated flux upon transfer from the growth medium to the experimental medium. Glucose (5 mM) and donor horse serum [1% (v/v) or lower] are the only additions required to maintain cell viability for prolonged periods (up to 3 hr). The flux measurement is initiated by the addition of the prewarmed radiotracer-containing solution (3..7 kBq/cm 3 SrRb; Amersham International, Amersham, England), and terminated by its rapid removal by suction via a water-driven pump and by repeated washing (five or six times, - 3 0 sec each time) in ice-cold tracer-free wash medium (usually the incubation medium). With cultured cells on plastic such washes achieve extremely rapid temperature reductions (~ 100 msec). Artifactual loss of isotope is thus low, but may be reduced by inclusion of transport inhibitors in the "stopping solution," e.g., HgCI2 for facilitated sugar transport. The adequacy of the wash protocol must be checked by the efficient clearance of an extracellular marker such as [~4C]- or [3H]inulin. The total time for measurement of undirectional uptake requires that the flux is undertaken in the linear portion of the uptake curve. For ~Rb a 5- or 10-rain influx period is usual. For other ions, e.g., 22 Na, extremely rapid influx determinations must be used (10 see); this is not easily performed since high ,2 F. V. Sepulveda and J. D. Pearson, J. Cell. Physiol. 118, 211 (1984). ,3 K. Matlin, D. F. Bainton, M. Pesonen, D. Louvard, N. Genty, K. Simons, J. CellBiol. 97, 627 0983).
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isotope-specific activities need to be used. It should be noted that determinations of influx of Na + in MDCK cells in some studies are made under conditions of net uptake in Na+-depleted cells. 14 Flux determinations are normalized to cell number/plate (most usual), protein, or DNA content using an aliquot of the extracted well. If flux is normalized by cell number determinations extraction of isotope is made with a trypsin- 2 m M EDTA solution to form a single-cell suspension as outlined above. An important control that is always performed is to test for nonspecific uptake of radiotracer on plastic Petri dishes in the absence of seeded cells. This is especially important for binding determinations. Unidirectional efflux is performed using a single-plate technique with continual replacement (every 3 min) of the external medium. This necessitates the preequilibration of the intracellular fluid with S6Rb by incubation in experimental medium containing 37 kBq/cm 3 S6Rb for 3 hr. Rate coefficients for S6Rb loss can be calculated from semilog plots of S6Rb loss against time. Measurements of net ion movements and of total ionic composition are made by washing the cell layers in ice-cold wash medium (usually isotonic choline chloride for Na + and K + determinations) and by final extraction in double glass-distilled water for 3 - 4 hr. Sodium ion and K + are directly determined using flame photometry. Determination of cell volumes in monolayer cultures is most conveniently measured using the technique of Kletzein et al? 5 Cell layers are incubated to equilibrium (60 rain) with 37 kBq/cm 3 of the nonmetabolizable sugar 3-O-methyl-D-[~4C]glucose as an intracellular space marker. It is important to check that no uphill transport occurs (as would occur in LLCPK t cells) and that there is no intracellular binding. Washing and extraction is carried out as described for K + fluxes. If the demonstration of the epithelial polarity of transport or binding is required, influx, etflux, and binding determinations are possible on epithelial layers grown upon permeant supports, using techniques identical in principle to those described. The filter support in this case will impose a serious "unstirred layer" for measurements across the basal surfaces. Preparation of Epithelial L a y e r s on P e r m e a n t Supports Uncoated Millipore (MF) Supports
Epithelial layers of MDCK cells have been successfully generated with pore sizes up to 2/~m. Generally the larger the pore size the slower the ~4M. J. Kindler,M. Taub, and M. H. Saier,J. CellBiol. 254, 11431 (1979). ~5R. F. Kletzein,M. W. Pariza,J. E. Becker,and V. R. Potter,Anal. Biochem. 69, 537 (1975).
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attainment of a confluent layer. We have not successfully generated epithelial layers in larger pore sizes without coating the support with collagen. Seeding is carried out by directly applying the cell suspension at high cell densities (2 × 105 cells/cm2 of permeable support) onto Millipore filters (2.5-cm diameter) (boiled in 100 cm 3 distilled water to remove the wetting agent) and held in presterilized Swinnex holders (Millipore Ltd., Harrow, England). The holder is incubated at 37* in an air/5% CO2 atmosphere for up to 1 hr to allow cell attachment. After this time the filter is removed from the holder, the layer is washed free of nonadherent cells, and the layer is finally floated in complete medium to which insulin is invariably added. After 3 days confluent epithelial layers are formed and we usually perform our experiments at this time. However, the medium may be changed every 3 days and the epithelial layers maintained. For routine experiments involving measurement of epithelial parameters we favor this method, as the epithelial layers may then be clamped directly into Ussing chambers in which edge effects are reduced in a conventional way. It should be stressed that edge effects exist even with epithelia grown in mini-Marbrook-type chambers, as epithelial cells are unlikely to form functional junctions with the vertical walls of the chamber. The morphology of MDCK cells certainly varies toward the outer edge of the cup under such conditions.
Collagen-CoatedMillipore Filters Collagen (5 mg/cm 3) [made from rat tails or purchased from Sigma (St. Louis, MO)] in 1% acetic acid is poured directly onto 2.5-cm Millipore filter disks contained in a 10-cm Petri dish and spread to give an even coverage. Gelation is achieved by exposure to ammonia vapor from 1 to 2 cm 3 of ammonia solution. The ammonia solution is removed and the filters allowed to dry. The collagen gel is then cross-linked by immersion in 4% glutaraldehyde solution for 60 min. Repeated washing with MEM ensures removal of any residual giutaraldehyde. Cell seeding is achieved by placing the collagen-coated filters in small (3 cm) dishes and proceeding as above.
Assessment of Confluency on Millipore Substrates Assessment of confluency in cell layers grown on Millipore filters is made by a conventional fixation (2% glutaraldehyde, staining with hemotoxylin) and dehydration schedule. Filters in absolute alcohol are then cleared in a 50:50 mixture of absolute alcohol and toluene, and finally in toluene. The filters are air dried and mounted on microscope slides in Canada balsam, which, since it has the same refractive index as the filter, renders it translucent. The epithelial layer may then be visualized directly.
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Growth of Epithelial Layers on Collagen-Coated Nylon Mesh Collagen gel may be used to form a translucent substrate in a nylon mesh support, as initially described by Cereijido et al. t6 Disks of nylon mesh (2.5 era; Simonyl N118, Henry Simon Textiles, Stockport Cheshire, England) are first cleaned by sonication in acetone (5 min), boiling in distilled water (10 min), and then washing in an ascending series of alcohols before being dried. They are then dipped in acetic acid-collagen solution and processed as are Millipore filters. The strength of the collagen gel was often sufficient such that the glutaraldehyde cross-linking step could be omitted. The omission of the glutaraldehyde fixation step facilitated microelectrode penetration through the collagen matrix. Final sterilization of the collagen/nylon substrates was achieved by short-wave UV irradiation (30 min). Storage of both collagen-covered Millipore filters and nylon/collagen matrices was possible in MEM for up to 1 month at 4". An alternative translucent substrate based on chitosan has been developed and shown to be perfectly adequate for the formation of MDCK epithelia.22 Growth in Marbrook- Type Chambers A variety of arrangements have been adopted to allow direct culture of epithelial layers in which separate access to each epithelial surface is achieved. Our initial method was to clamp Millipore filters directly into commercially available (Hendley Engineering Co., Loughton, Essex, England) nylon mini-Marbrook chambers. 17 These chambers consist of two portions to which the filter directly clips. They may be sterilized preassembled and have small feet to allow direct access of medium to the basal surfaces of the developing epithelium. Seeding is achieved by direct addition of the concentrated cell suspension to the chamber outlined above, except that fresh growth medium is finally added to both the upper chamber ( 1.5 cm a) and the 3-cm Petri (3 cm a) dish in which the mini-Marbrook chamber is placed. During growth the upper chamber often becomes acidified before the lower chamber, indicating functional (H +) transport. The great advantage of the mini-Marbrook arrangement is for longer term experiments in which epithelial cultures can be monitored continually using salt bridges cleaned by alcohol wipes, and for experiments in which access to both epithelial surfaces is required for collection of secreted products, for biochemical labeling experiments, or for transepithelial cell migration studies. 16 M. Cereijido, E. S. Robbins, W. J. Dolan, C. A. Rotunno, and D. D. Sabatini, J. Cell Biol. 77, 853 (1978). '~ J. C. W. Richardson and N. L. Simmons, FEBSLett. 105, 201 (1979).
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More recently several different designs have been introduced encompassing similar design features to those existing in the commercial miniMarbrook chambers.~8 In addition, different sizes of presterilized chambers consisting of membrane filter glued to a polystyrene body are now available [e.g., Millicell-HA; a transparent membrane filter (Biopore, MillicellCM) allows direct microscopy of growing epithelial layers. Such transparent membranes need to be precoated with a biological matrix such as collagen; our own experience is that lower epithelial resistance values are achieved with Millicell-CM compared with Millicell-HA.] A novel adaptation of this idea was developed by the Simons group in Heidelburg. ~9The Millipore millititer HA 96-well plate for microfiltration can be used to produce a large number of identical epithelial layers suitable for rapid screening of the effects of agents such as monoclonal antibodies (or other bioactive reagents) on epithelial junction formation. The impermeable backing is first stripped from the 96-well plate, and the sterile packaging bubble can be used as the bottom chamber. Cell seeding is achieved, exactly as described above. Our experience is that entirely adequate epithelial layers may be generated, though the electrical resistance is of a somewhat lower magnitude. Measurement of Transepithelial Electrical Properties (Resistance, Short-Circuit Current, Dilution, and Biionic Potential Differences (pd) A detailed and basic discussion of this topic, including construction, calibration, and use of reversible electrodes and salt bridges, is given in an excellent review by Watlington et al. 2° As previously mentioned, epithelial layers may be mounted directly using large areas and silicone seals to reduce edge effects, 21 o r whole mini-Marbrook chambers may be clamped directly into specially adapted chambers. For the purpose of the present chapter it is worth stressing the importance of the correct geometry needed with respect to the measuring and current-passing Ringer bridges, when applied to the p.d. measurement of the electrical resistance of "leaky" epithelial layers, and of the necessity of making an appropriate correction for the series resistance afforded by the solution resistor present between the potential-sensing electrodes. This point is also of practical importance 18 R. E. Steele, A. S. Preston, J. P. Johnson, and J. S. Handler, Am. J. Physiol. 251, C136 0986). ,9 D. Gumbiner and K. Simons, J. Cell Biol. 102, 457 0986). 2o C. O. Watlington, J. C. Smith, and E. G. Huf, Exp. Physiol. Biochem. 3, 49 (1970). 21 N. L. Simmons, C. D. A. Brown, and E. L. Rug& Fed. Proc., Fed. Am. Soc. Exp. Biol. 43, 2225 0984).
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when short circuiting the epithelial layer. In standard Ussing chambers made of Perspex, drilled guide holes for the potential and current-passing agar bridges are present, ensuring that no change in the distance between the potential-sensing electrodes occurs on chamber assembly/disassembly, and that the current-passing bridges are placed sufficiently distant from the epithelial layer to ensure an even current density (the rule of thumb is two times the radius of the epithelial layer). For accurate determination of epithelial properties, it is simply not sufficient to dip the agar bridges in the bathing solutions, though this may be adequate for rapid screening. Often the DC electrical resistance of the epithelium might not be the most convenient or reliable method for determining the functional presence of an epithelial layer. If a significant paracellular pathway for ions is present with ionic mobilities differing from free solution, a more convenient method is to elicit a dilution or biionic pd. For MDCK epithelial layers this can be achieved by replacing the basal bathing solution for one in which the NaC1 is replaced in an equimolar fashion by choline chloride) 6 The resulting concentration gradient for Na + and the cationic permselectivity of the paracellular pathway will render the basal bathing solution up to 45 m V electropositive with respect to the apical bathing solution. Replacement of the basal bathing solution is recommended as the epithelial layer is less subject to damage from solution turbulence under these conditions. M e a s u r e m e n t of Bidirectional Transepithelial Solute Fluxes Simultaneous measurement of bidirectional transepitbelial fluxes using 3H-or 14C-labeled solutes or 22Na and UNa is recommended whenever possible in order to eliminate time-dependent variation and betweenmonolayer variation. Isotopes are added to both bathing solutions (volume = 8 crn 3) and after an initial 30-rain flux period, 1-cm 3 samples of each chamber are taken. Two further 1-cm 3 samples at 60 and 90 rain are taken and fluxes averaged over these two 30-rain periods. Flux is calculated from the rate of tracer appearance on the contralateral side, to which radiotracer was added. Isotope equilibration should not exceed 1 -2%, and the specific activities used should allow increments of 5 X 102 Bq/cm 3 in the contralateral chamber.
22 p. Popowicz, J. Kurzyca, B. Dolinska, and J. Popowicz, Biomed. Biochim. Acta 44, 1329 (1985)
